Defects in the mitochondrion, the energy-producing unit of the cell, lead to a wide range neural and muscular diseases caused by decreased energy production, free radical damage, and perturbations to apoptotic pathways. The protein import pathways of the mitochondrion mediate the import and assembly of proteins from the cytosol. The X-linked disease Mohr-Tranebjaerg syndrome or deafness-dystonia syndrome is caused by a specific defect in the import of inner membrane proteins. The goal of this proposal is to investigate the mechanism of protein import into the mitochondrion in the experimental model, the budding yeast Saccharomyces cerevisiae and to extend our studies into mammalian systems. The TIM22 import pathway and the Erv1/Mia40 oxidative folding pathway will be targeted. The objective of this research is to define the molecule mechanisms of protein import with a combined biochemical, biophysical, and genetic approach. Specifically, the mechanism by Mia40 and Erv1 mediate protein import and disulfide bond assembly in the intermembrane space will be elucidated. In addition, a chemical- genetic approach will be utilized to identify small molecule effectors that may modulate the TIM 22 import pathway, with the long-term goal of developing therapeutics for deafness-dystonia syndrome and other mitochondrial disorders, as well as the Mia40/Erv1 pathway with a long- term goal of understanding the molecular basis of a new mitochondrial disease caused by mutations in Erv1. The proposed project will expand fundamental knowledge about the mechanism of protein insertion into the mitochondrial inner membrane, extending present studies that have focused generally on the mechanism by which proteins reach soluble compartments of the mitochondrion. Importantly, this research will generate new chemical probes for investigating mitochondrial assembly in yeast, cultured mammalian cells, and animal models such as fly, worms, and zebrafish that will be available to the mitochondrial research community. This research will impact public health because these mechanistic studies will provide insight into how defects in mitochondrial biogenesis lead to diseases such as deafness- dystonia syndrome, Friedreich's ataxia, and Parkinson's and Alzheimer's disease, which are caused by mitochondrial dysfunction. The ultimate goal of this research is to use our models in a chemical-genetic approach to identify small molecule effectors, which in the long-term may lay the groundwork for developing new tools to understand how mitochondrial defects lead to human diseases and new therapeutic approaches to develop drugs that will modulate mitochondrial function.